Some mammalian reproductive cells that are extremely sensitive to low temperature, such as bovine oocytes and embryos, can be successfully cryopreserved by vitrification at ultra-rapid cooling rates. U.S. Pat. No. 6,500,608 to Forest et al. discloses a method to cryopreserve cells that are highly sensitive to low temperature by vitrification using a loop as a transfer instrument. U.S. Pat. No. 6,982,172 to Yang et al. discloses a method to vitrify oocytes or embryos at ultra-rapid cooling rate by the direct contact of small volume of vitrification solution containing bovine oocytes to the surface of a very cold (−150˜180° C.) solid surface with good thermal conductivity. However, cryopreservation of cells with very high levels of intracellular lipids, such as produced porcine embryos, by these approaches does not yield good results.
Intracellular lipid levels are inversely related to cryosurvival of some animal cells. For example, the exceedingly temperature sensitive nature of some domestic animal oocytes and embryos has been proven to be due to their very high level of intracellular lipids. A reduction in the content of intracellular lipids can significantly improve their survival after cryopreservation, a phenomenon that has been well documented in porcine embryos.
Reduction of intracellular lipid contents in porcine embryos is usually accomplished by employing mechanical delipation. The intracellular lipids in porcine embryos are first polarized to one side of embryos by ultracentrifugation. Subsequently, the polarized lipid droplets are removed by micromanipulation. Thus far, this approach has resulted in the best cryosurvival in porcine embryos generated both in vivo and in vitro. However, due to the extensive resources needed, this approach offers very limited practical value. This approach also significantly increases the chance of pathogen transmission because of the damaged zona pellucida after micromanipulation. In addition, it is also extremely labor-intensive and time-consuming.
U.S. Pat. No. 6,503,698 to Dobrinsky and Nagashima discloses a modified mechanical delipation approach applied to the cryopreservation of porcine embryos derived in vivo. In this method, the intracellular lipid droplets are polarized to one side of embryos through centrifugation at a very high speed (13,000 g) and the embryos are then cryopreserved without removal of the polarized lipid droplets. This approach results in a reasonable survival of porcine embryos derived in vivo. However, after cryopreservation, the polarized lipid droplets lose their ability to redistribute into the cytoplasm which may have a detrimental effect on the subsequent development of porcine embryos.